Highly active antiretroviral therapy (HAART) has had a major impact on the acquired immunodeficiency syndrome (AIDS) epidemic in industrially advanced nations. However, eradication of human immunodeficiency virus type 1 (HIV-1) does not appear to be currently possible, in part due to the viral reservoirs remaining in blood and infected tissues. Moreover, a number of challenges have been encountered, which include various adverse effects, only partial and limited immunologic restorations achieved, and the occurrence of various cancers as consequences of survival elongation with HAART. Moreover, such limitations of HAART are exacerbated by the development of drug-resistant HIV-1 variants. Thus, the identification of new classes of antiretroviral drugs that have a unique mechanism(s) of action and produce no or minimal adverse effects remains an important therapeutic objective. Dimerization of HIV-1 protease subunits is an essential process for the acquisition of proteolytic activity of HIV-1 protease, which plays a critical role in the maturation and replication of the virus. Thus inhibition of protease dimerization (PD) by chemical reagents is likely to abolish proteolytic activity and inhibit HIV-1 replication. However, to develop HIV-1 protease dimerization inhibitors, a better understanding of the nature and dynamics of protease dimerization is crucial. The monomer subunits are connected by polar and non-polar interactions to form the dimer. Hydrophobicity of Leu89, Leu90, Ile93 and several other residues have been considered important in the folding of a protease monomer as well as in dimer stabilization. For a systematic analysis of the conserved network of hydrogen bonds, termed firemans grip, Strisovsky et al. have mutated the active site Thr26 to a Ser, Cys, or Ala and have shown that T26A substitution reduced protease dimer stability, thus virtually nullifying the proteolytic activity of protease. Indeed, in our present study, T26A substitution effectively disrupted protease dimerization. The flexibility of monomeric and dimeric HIV-1 protease and the feasibility of a stable protease monomer have also been studied by computational simulation. There are four anti-parallel beta-sheets involving the N- and C- termini of both monomer subunits and they contribute close to 75% of the dimerization energy, explaining at least in part why darunavir (DRV) failed to dissociate mature protease dimer. The termini interface has been explored as a dimerization inhibition target by several groups. We have also recently reported that certain peptides containing the dimer interface sequences amino acids 1-5 and amino acids 95-99 blocked HIV-1 infectivity and replication. However, to the best of our knowledge, no evidence of direct dimerization inhibition by such compounds has been yet documented. In the present study, we developed an intermolecular fluorescence resonance energy transfer (FRET)-based HIV-1-expression assay that employed cyan and yellow fluorescent protein-tagged HIV-1 protease monomers. Using this assay, we identified a group of non-peptidyl small molecule inhibitors of HIV-1 protease dimerization. These inhibitors, including the recently approved protease inhibitor (PI) darunavir (DRV) as well as two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 microM and blocked HIV-1 replication in vitro with IC50 values of 0.0002-0.48 microM. These agents also inhibited the proteolytic activity of mature HIV-1 protease. Another PI, tipranavir (TPV), active against HIV-1 variants resistant to multiple PIs, also blocked protease dimerization, although all other existing FDA-approved anti-HIV-1 drugs examined in the present study failed to block the dimerization. The inhibition of HIV-1 protease dimerization by non-peptidyl small molecule agents represents a unique mechanism of HIV-1 intervention and the dually functional inhibitors reported here might serve as potential candidates as a new class of therapeutic agents for HIV-1 infection and AIDS. The present data that have been obtained in this project should not only help design and examine agents that potentially inhibit HIV-1 protease dimerization, but should also produce new insights into the process and dynamics of HIV-1 protease dimerization per se.